The present invention relates to a protein involved in long-term survival of cranial nerve cell, to DNA encoding the protein, and to uses thereof. More particularly, the present invention relates to human-derived bradeion protein or derivatives thereof, to DNA encoding the protein or the derivatives thereof, to a vector containing the DNA, to a host cell transformed or transfected with the vector, to an antibody immunologically reactive with the protein or the derivatives thereof, and to uses of the DNA or the antibody for detecting a cancer.
Cranial nerve cells (neurons) are main elements for controlling survival of higher order animals. Once the neurons are produced, they do not divide at all and only gradually exfoliate or go through necrosis. Exfoliation of the neurons occurs in the normal state but is particularly accelerated by genetic diseases, brain ischemia, or status epilepticus, or under conditions of poor nutrition and low oxygen. Some disorders of cranial nerves associated with aging (e.g., dementia) result from deficiency of an absolute amount of functional neurons caused by accumulation of exfoliated neurons. Thus, the monitoring and control of the exfoliation, as well as regeneration of the functions of neurons, are the most demanding subject to be solved among the aging problems.
Cranial nerve cells do not divide at all after the induction phase of differentiation in the process of development, and maintain their functions or is accompanied by gradual deterioration of their functions until the end of the life-time of the individual. They are presumed to have specific division-interrupting and function-maintaining mechanisms although these mechanisms have not yet been clarified. In the central nervous system, there exist numbers of unknown proteins and signaling substances, particularly stimulating substances and receptors thereof involved in brain-specific signal transduction, but their details are yet unclear.
Numbers of researches have been conducted worldwide on such an important element that controls the survival of the cranial nerve cells. However, none of the elements was clarified in the substance or molecule level, and, prior to everything, it was necessary to develop techniques for analysis. Recently, the group of Dr. Masashi Yanagisawa and his colleagues of the University of Texas, Medical Research Center (authorized by the Howard Hughes Foundation) has succeeded in developing a technique for randomly screening neuropeptides and receptors thereof by using cultured cells, and they have found a substance (named orexin) that directly binds to and stimulates the aperitive center in the hypothalamus, and identified functions of the substance""s receptor (Cell, 92, 573-585, 1998). However, such a systematic screening of substances is rare, and currently, stimulating factors involved in brain-specific signal transduction and receptors thereof are not yet fully clarified.
Under such circumstances, the present inventors have now constructed an improved expression gene (cDNA) library, developed a systematic screening technique, and succeeded in extraction and selection of genes specific for cranial nerve cells, thereby accomplishing the present invention.
Thus, one object of the present invention is to provide a bradeion protein involved in long-term survival of cranial nerve cells, and DNA coding for the bradeion protein.
Another object of the present invention is to provide a vector containing the above-mentioned DNA, and a host cell transformed or transfected with the vector.
Still another object of the present invention is to use the DNA or an antibody to the protein for detecting cancers.
The present invention provides a human-derived bradeion protein, which has the following properties:
(i) it is a transmembranous protein;
(ii) it has a structure characteristic of growth hormone and cytokine receptors (even in a structure of its transmembranous portion) when its structure is determined by a hydrophobicity analysis according to Kyte-Doolittle method;
(iii) it is expressed at a high level in the human adult brain, in less amount in the heart, while it is not expressed in other adult organs or fetus;
(iv) it induces programmed cell death (apoptosis) when over-expressed in cultured human cell lines;
(v) it induces termination of cell division and aging when over-expressed in cultured human normal cells;
(vi) it is located in cytoplasm, and forms an intracellular aggregate when overexpressed; and
(vii) besides human adult neurons, it is specifically expressed in a human colorectal cancer cell line or in a skin cancer cell line,
or an analogue thereof.
The proteins of the invention include Bradeion xcex1 and Bradeion xcex2 proteins having the amino acid sequences shown in SEQ ID NOS:2 and 4, respectively. These proteins are not the consequence of alternative splicing, but coded in the adjacent area (chromosome 17q23) of human genome. In addition to the above-described properties, Bradeion xcex1 induces programmed cell death when DNA coding for Bradeion xcex1 or an analogue thereof is introduced into a cultured cancer cell.
The term xe2x80x9canaloguexe2x80x9d as used herein refers to a protein that has properties substantially equivalent to those of the human-derived bradeion proteins (for example, at least the properties of (i), (ii), (iii), (vi) and (vii)), or a protein having an amino acid sequence with deletions, substitutions or additions of at least one amino acid residue in the amino acid sequence shown in SEQ ID NO:2 or 4, or a protein having an amino acid sequence that is substantially the same as that shown in SEQ ID NO:2 or 4. Preferably, the analogue of the invention has at least 90%, preferably at least 95%, more preferably at least 97% homology with the amino acid sequence of SEQ ID NO:2 or 4. The analogue of the invention also includes human bradeion proteins modified or mutated in the amino acid level, and bradeion proteins from non-human mammals having properties substantially equivalent to those from humans. Only Bradeion xcex2 (similar to the human-derived Bradeion xcex2) was found, for example, in a mouse brain and its homology with the human Bradeion xcex2 was 94%. The analogues of the invention may be obtained through DNA recombinant techniques by artificial modifications (e.g., site-directed mutagenesis), as long as the original bioactivity of the human bradeion is not impaired. The analogues of the invention may or may not contain a sugar chain, or they may be chemically modified with an aqueous polymer such as polyethylene glycol.
Moreover, the present invention provides DNAs comprising nucleotide sequences coding for the above-defined bradeion proteins or analogues thereof, and fragments of the DNAs.
Specific examples of such DNAs or fragments thereof include: DNA comprising the sequence of the nucleotides 129-1943 of SEQ ID NO:1 (i.e., DNA encoding human Bradeion xcex1); DNA fragments having at least 15, preferably at least 20, more preferably at least 30 consecutive nucleotides derived from the nucleotides 129-1943 of SEQ ID NO:1; DNA comprising the full-length nucleotide sequence shown in SEQ ID NO:1; DNA comprising the sequence of the nucleotides 129-1562 of SEQ ID NO:3 (i.e., DNA encoding human Bradeion xcex2); DNA fragments having at least 15, preferably at least 20, more preferably at least 30 consecutive nucleotides derived from the nucleotides 129-1562 of SEQ ID NO:3; and DNA comprising the full-length nucleotide sequence shown in SEQ ID NO:3. In addition, DNA that can hybridize with one of the above-mentioned DNAs under stringent conditions is also encompassed in the present invention. The stringent conditions as mentioned herein refer to such conditions that allow hybridization only when there is at least 90% homology, preferably at least 95% homology, more preferably at least 97% homology with the nucleotide sequence shown in SEQ ID NO:1 (positions 129-1943) or SEQ ID NO:3 (positions 129-1562). Generally, such conditions allow hybridization at a temperature that is lower by about 5xc2x0 C.-30xc2x0 C., preferably by about 10xc2x0 C.-25xc2x0 C. than the melting temperature (Tm) of the complete hybrid. Stringent conditions that may be used are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), esp. xe2x80x9cConditions for Hybridization of Oligonucleotide Probesxe2x80x9d. The DNA and fragments thereof according to the present invention can be used not only for the expression of the bradeion proteins but also as a probe for hybridization or as a primer for PCR.
The present invention further provides vectors containing DNAs coding for bradeion proteins or analogues thereof, or fragments of the DNAS. The vector usually contains a promoter that is capable of operably expressing the DNAs. In addition to the promoter, the vector may contain at least one other element such as an origin of replication, an enhancer, a ribosome-binding site, a transcription termination factor (terminator), a selective marker, an RNA splicing site, or a polyadenylation signal.
The present invention further provides a host cell that has been transformed or transfected with such a vector. The host cell may be a prokaryotic or eukaryotic cell, preferably an eukaryotic cell, more preferably a mammalian cell such as a human cell line.
The present invention also provides an antibody that is immunologically reactive with the above-defined bradeions or analogues thereof. The antibody is preferably one that can specifically immuno-react with the bradeion proteins or analogues thereof, and is a polyclonal or monoclonal antibody.
The invention further provides a method for detecting a cancer, comprising detecting the cancer by using the above-defined DNAs or fragments thereof or the above-defined antibodies as tumor markers. Herein, the cancer includes, but is not limited to, a human colorectal cancer and a human skin cancer. The detection can be conducted by hybridization or immunoassay.
This specification includes all or part of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 10-325380, which is a priority document of the present application.